Methods for the characterization of phosphatase-stabilized 2-5A-antisense chimeras.
We have developed chromatographic and spectrophotometric assays for determining the degree of thiolation in phosphatase-resistant 5'-monothiophosphate-capped 2-5A-antisense chimeras. Concomitantly, we have explored the reactivity of this 5'-monophosphorothioate moiety with reporter reagents such as 5-iodoacetomidofluorescein and 5,5'-dithiobis(2-nitrobenzoic acid). On the basis of these reactions, analyses for 5'-monothiophosphate-functionalized 2-5A-antisense chimeras were made possible. Kinetic experiments demonstrated that oligonucleotide backbone negative charge could retard mixed disulfide formation in the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with 5'-monothiophosphorylated 2-5A-antisense chimeras.
Player
M R
MR
Section on Biomedical Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0805, USA.
Xiao
W
W
Cramer
H
H
Torrence
P F
PF
D000227:Adenine Nucleotides / Q000737:chemistry*; D000998:Antiviral Agents / Q000737:chemistry*; D002072:Butylene Glycols / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D009843:Oligoribonucleotides / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation
D000227:Adenine Nucleotides; D000998:Antiviral Agents; D002072:Butylene Glycols; D005452:Fluoresceins; D016376:Oligonucleotides, Antisense; D009843:Oligoribonucleotides; D010710:Phosphates; C023505:2',5'-oligoadenylate; C015296:5-iodoacetamidofluorescein; C039681:1,4-butanediol; D004228:Dithionitrobenzoic Acid; D010744:Phosphoric Monoester Hydrolases; C035638:thiophosphoric acid